Ablation of the oncogenic transcription factor ERG by deubiquitinase inhibition in prostate cancer - PubMed (original) (raw)

. 2014 Mar 18;111(11):4251-6.

doi: 10.1073/pnas.1322198111. Epub 2014 Mar 3.

Rahul K Kollipara, Nishi Srivastava, Rui Li, Preethi Ravindranathan, Elizabeth Hernandez, Eva Freeman, Caroline G Humphries, Payal Kapur, Yair Lotan, Ladan Fazli, Martin E Gleave, Stephen R Plymate, Ganesh V Raj, Jer-Tsong Hsieh, Ralf Kittler

Affiliations

Ablation of the oncogenic transcription factor ERG by deubiquitinase inhibition in prostate cancer

Shan Wang et al. Proc Natl Acad Sci U S A. 2014.

Abstract

The transcription factor E-twenty-six related gene (ERG), which is overexpressed through gene fusion with the androgen-responsive gene transmembrane protease, serine 2 (TMPRSS2) in ∼40% of prostate tumors, is a key driver of prostate carcinogenesis. Ablation of ERG would disrupt a key oncogenic transcriptional circuit and could be a promising therapeutic strategy for prostate cancer treatment. Here, we show that ubiquitin-specific peptidase 9, X-linked (USP9X), a deubiquitinase enzyme, binds ERG in VCaP prostate cancer cells expressing TMPRSS2-ERG and deubiquitinates ERG in vitro. USP9X knockdown resulted in increased levels of ubiquitinated ERG and was coupled with depletion of ERG. Treatment with the USP9X inhibitor WP1130 resulted in ERG degradation both in vivo and in vitro, impaired the expression of genes enriched in ERG and prostate cancer relevant gene signatures in microarray analyses, and inhibited growth of ERG-positive tumors in three mouse xenograft models. Thus, we identified USP9X as a potential therapeutic target in prostate cancer cells and established WP1130 as a lead compound for the development of ERG-depleting drugs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Identification of USP9X as an ERG-binding protein. (A and B) Detection of ERG-binding proteins by pulldown with recombinant GST-ERG (A) or immunoprecipitation with an antibody against ERG (B) from VCaP whole cell extract. Proteins were separated by SDS/PAGE and detected by Coomassie blue staining. The asterisk indicates IgG heavy chain. (C) Coimmunoprecipitation of USP9X with endogenous ERG in VCaP cells with an antibody directed against ERG. Immunoblotting was performed with antibodies against ERG and USP9X. (D) USP9X protein expression in benign prostate (n = 37), ERG-negative (n = 63), and ERG-positive (n = 34) prostate cancer specimens (P values, Student's t test).

Fig. 2.

Fig. 2.

USP9X stabilizes ERG by deubiquitination, which is inhibited by WP1130. (A) USP9X knockdown increases ERG ubiquitination. HeLa cells were transfected with siRNAs against USP9X or control siRNA (siNT); after 72 h, these cells were cotransfected with ERG-V5 and HA-ubiquitin expression constructs. After 24 h, ERG-V5 was immunoprecipitated, and immunoblotting was performed with antibodies against V5 and HA to detect ubiquitinated ERG (ub-ERG), or with antibodies against USP9X and GAPDH for input to monitor the efficiency of the USP9X knockdown. The asterisk indicates monoubiquitinated ERG (65 kDa). (B) USP9X deubiquitinates ERG in vitro. Ubiquitinated ERG-V5 was incubated with wild-type USP9X-FLAG, mutant USP9X-mut-FLAG, or mock (control), followed by immunoblotting. (C) USP9X knockdown decreases ERG levels in VCaP cells. VCaP cells were transfected with siRNAs directed against USP9X or siNT. (D) WP1130 increases the levels of ubiquitinated ERG. HeLa cells expressing ERG-V5 and HA-ubiquitin were treated with 5 µM WP1130 for 0–4 h before ERG-V5 immunoprecipitation. (E) WP1130 causes ERG depletion in VCaP cells. VCaP cells were treated with WP1130 for 24 h. Quantification of triplicate immunoblot analysis for C and E is shown in

SI Appendix, Table S7

.

Fig. 3.

Fig. 3.

Effects of WP1130 treatment on prostate cancer cells. (A_–_F) Prostate tumor explants respond to WP1130 treatment. Prostate tumor ex vivo cultures are generated from freshly resected tumor specimens and grown on gelatin sponges in culture (A). ERG IHC staining in ERG+ prostate tumor explants following incubation for 24 h in either the presence of vehicle (DMSO) (B) or 10 µM WP1130 (C). (Scale bar: 100 µm.) Quantification of ERG levels in ERG+ tumors (n = 5) (D). WP1130 treatment strongly inhibits proliferation in ERG_+_ tumors (n = 5) (E) but not in ERG− tumors (n = 7) (F) as indicated by Ki-67 staining. Error bars represent SD. (G_–_L) WP1130 treatment reduces DNA damage. VCaP cells were treated for 48 h with DMSO (G) or 2 µM WP1130 (H) before immunofluorescence analysis of ERG, γH2AX, and DNA (DAPI). Quantification of γH2AX after WP1130/control treatment (I), RNAi knockdown of ERG and USP9X (J) in VCaP cells, or WP1130/control treatment in PC3 (K) and Du-145 (L) cells. Error bars represent SD (P values, Student's t test).

Fig. 4.

Fig. 4.

Effects of WP1130 on ERG levels and tumor growth in vivo. (A and B) WP1130 inhibits the growth of ERG-overexpressing tumors in mice. Mice xenografted with ERG-overexpressing VCaP cells (A) or 22Rv1-ERG and 22Rv1-vector cells (B) were treated upon appearance of tumors with 40 mg/kg WP1130 or vehicle (i.p.) every other day beginning on day 27 (A) or 12 (B) after cell injection. Error bars represent SEM. (C and D) WP1130 causes ERG depletion in vivo. Tumors were obtained from VCaP (C) and 22Rv1-ERG (D) tumor-bearing mice 24 h after injection with vehicle or WP1130 (40 mg/kg). Immunoblotting was performed with antibodies against ERG, GAPDH, and COX IV (human protein-specific antibody). (E) Appearance of xenograft VCaP, 22Rv1-ERG, and 22Rv1-vector tumors at the end of the treatment course with vehicle or WP1130 (40 mg/kg). Treatment with WP1130 was not associated with any apparent toxicity (e.g., weight loss or behavioral changes).

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